The technical field of this invention is a particulate chitosan core matrix for viable cells encapsulated in vehicles intended for implantation into an individual.
A number of substances have been employed as core material for the encapsulation of cells in microspheres and macrocapsules. Typically, the core material is formed in a gel in which the cells are imbedded. The gelled core may then be further encapsulated in a semipermeable membrane to form an implantable vehicle.
The ideal core material would provide a physical support for the cells to keep them evenly dispersed throughout the core. If cells tend to clump within the core, the cells in the middle of the clump may be deprived of oxygen and other nutrients and become necrotic. The core matrix should also be sufficiently permeable to substances secreted by the cells so that a therapeutic substance can diffuse out of the core and into the tissue or blood stream of the recipient of the implanted vehicle. If proliferation or differentiation of cells within the core is desired, the core matrix should also provide a physio-chemical environment which promotes those cellular functions.
One commonly employed core material is the anionic polysaccharide gum, sodium alginate, as disclosed in U.S. Pat. Nos. 4,352,883 (Lim, F.), U.S. Pat. No. 4,689,293 (Goosen, M. F. A., et al.), U.S. Pat. No. 4,806,355 (Goosen, M. F. A., et al.), U.S. Pat. No. 4,789,550 (Hommel, M., et al.), U.S. Pat. No. 4,409,331 (Lim, F.), and U.S. Pat. No. 4,902,295 (Walthall, B. J., et al.).
Other core materials include collagen (U.S. Pat No 4,495,288, Jarvis, A. P. et al.), agar, agarose, fibrinogen (U.S. Pat. No. 4,647,536, Mosbach, K., et al.), and fibronectin or laminin (U.S. Pat. No. 4,902,295, Walthall, B. J., et al.).
The core material of the present invention is chitosan, which is a derivative of chitin. Chitin is the major component of the shells of shrimp and crab, and is produced commercially as a by-product of the shellfish industry.
Chitin is a linear polymer comprised of 2-acetylamino-D-glucose units. The term "chitosan" refers to a family of polymers, derived from chitin, that have been partially deacetylated to provide sufficient free amino groups to render the polymer soluble in selected aqueous acid systems (Filar., L. J., et al., Hercules Research Center Contribution No. 1697, Wilmington, Del.). Chitosan is commercially available in varying degrees of deacetylation ranging upwards from less than 75%. The degree of solubility of chitosan with a given degree of deacetylation depends on polymer molecular weight, temperature, and concentration and nature of the acid solvent (Filar, L. J., et al. supra).
Chitosan was reportedly used experimentally as a dura mater substitute (Muzzarelli, R., et al., 1988 Biomaterials 9:247-252). The dura mater is the sheet of collagenous connective tissue which encases the brain within the skull. The success of chitosan in this experimental paradigm was attributed to the fact that its structural characteristics are similar to the glycosaminoglycan components of naturally occurring extra-cellular matrix. In the presence of chitosan, fibroblasts and mesenchymal vascular cells in the surrounding tissue were stimulated to migrate, proliferate, and differentiate. These cellular activities are essential components of wound healing and tissue-rebuilding. Chitosan has also been reported to be effective in bone-repair and as a suture material (Sapelli. P. L., et al. 1986 in Chitin in Nature and Technology Eds. R. A. A. Muzzarelli, et al., Plenum Press, N.Y.: Nakajuma, M., et al. 1986 Jpn J Surg 16:418-424).
A feeding solution containing liquid chitosan was added to growing myocyte cultures and reportedly enabled the three-dimensional growth of the myocytes in culture plates (Malette, W. G., et al. U.S. Pat. No. 4,605,623). A chitosan/collagen material was reportedly effective in promoting cell substrate adhesion and proliferation in culture (Miyata, T., et al. EP 0318286).
Chitosan has been used to form a depot for the sustained release of pharmacologically active macromolecules such as hormones, enzymes, and protein antigens (Cardinal, J. R., et al. U.S. Pat. No. 4,895,724).
Processes have been disclosed for using chitosan to encapsulate living cells (Daly, M M., et al. U.S. Pat. No. 4,808,707; Rha, C.-K., et al., U.S. Pat. No. 4,744,933; Rha, C.-K., et al., U.S. Pat. No. 4,749,620; Schroder, U., U.S. Pat. No.4,713,249; Jarvis, A. P., U.S. Pat. No. 4,803,168). These processes are based on the cross-linking of the cationic free amino groups of chitosan via ionic bonds with anionic species such as phosphate ions or anionic polymers such as alginate. The terms "cross-linking", "cross-linked", etc. as defined herein mean ionic bonds or bridges between distinct chitosan chains or between distinct regions of a single chain.
The capsules of Rha are formed by dropping cationic chitosan solution into anionic alginate solution. The positively charged free amino groups of the chitosan polymers on the surface of the chitosan droplet are attracted to the negatively charged carboxylate groups of the alginate polymers, forming cross-links at the interface between the chitosan droplet and the alginate solution. The interfacially cross-linked chitosan-alginate forms a membrane enclosing a liquid chitosan core. Daly uses similarly interfacially cross-linked chitosan in which the composition of the alginate used is varied in order to alter the permeability properties of the capsule membrane. The microcapsules of Jarvis are formed by cross-linking the core chitosan polymers by the addition of divalent or multivalent anions, then forming permanent interfacial cross-links at the outer surface to a polymer having plural anionic groups, such as polyaspartic or polyglutamic acid. The inner core of the Jarvis capsules may be used in the cross-linked state, or may be reliquefied by the addition of low molecular weight cations. In all of these cases, the principal function of chitosan is to serve as one half of a pair of charged polymers which form interfacial cross-links resulting in the formation of the capsule membrane or wall.
The chitosan matrices of Schroder, supra, are formed through crystallization of the carbohydrate polymer to form a polymeric lattice. The capsules thus formed are used to deliver non-living biologically active substances. The methods employed for crystallization are generally considered too harsh for the encapsulation of living cells.
A major commercial producer of chitosan, Protan Laboratories, has published several protocols for the immobilization of cells within chitosan gels. These methods all employ ionotropic gelation (i.e. cross-linking) of chitosan by combining the chitosan/cell solution with anionic species. Proposed anions include polyphosphates, alginate, carrageenan, and fatty acids with sulfate moieties. (Technical bulletin: Chitosans for Cell Immobilization, Protan Laboratories, Redmond, Wash.). Cross-linked chitosan, however, has not been used as a core matrix for viable cell encapsulation in capsules with thermoplastic jackets. Nor has non cross-linked particulate chitosan ever been used as a core matrix for living cell encapsulation in encapsulation devices.
An object of this invention, therefore, is to provide a novel non cross-linked particulate chitosan core matrix for living cell encapsulation in encapsulation devices.
Another object of this invention is to provide a particulate chitosan core matrix in capsules with thermoplastic jackets where formation of the capsule wall is not dependent upon the presence of the chitosan matrix (e.g. through interfacial cross-linking) so that the properties of either the jacket or the matrix may be varied without concern for effects on each other.
Additional objects and features of the invention will be apparent to those skilled in the art from the following detailed description and appended claims when taken in conjunction with the figures.